Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in theLotus japonicus–Mesorhizobium lotisymbiosis

Mitsutaka Fukudome,Maki Nagata,Ken-Ichi Osuki,Jens Stougaard,Manuel Becana,Toshiki Uchiumi,Tomohiro Kado,Laura Calvo-Begueria,Ei-Ichi Murakami,Ken-Ichi Kucho,Maria Carmen Rubio,Niels Sandal

doi:10.1093/jxb/erw290

Abstract

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels.

Highlights

Leghemoglobins are hemeproteins with a relatively high O2 affinity which are found exclusively in legume nodules (Appleby, 1984; Becana and Klucas, 1992; Smagghe et al, 2009)
Four mutant homozygous lines were established by self-pollination and designated as P59S, A102V, E127K, and E136K according to the amino acid substitutions and their positions
All of them showed lower plant length than the WT, but only the A102V and E127K lines were selected for further studies because A102 is conserved and close to H104, which is important for assembling the heme moiety (Andersson et al, 1996), and because E127 is highly conserved among class 1 plant

Summary

Introduction

Leghemoglobins are hemeproteins with a relatively high O2 affinity which are found exclusively in legume nodules (Appleby, 1984; Becana and Klucas, 1992; Smagghe et al, 2009). They transport and deliver O2 to the symbiosomes at a steady but low concentration to avoid nitrogenase inactivation in bacteroids (Appleby, 1984) and are essential for symbiotic N2 fixation (Ott et al, 2005).

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Conclusion